A novel allosteric inhibitor of the uridine diphosphate N-acetylglucosamine pyrophosphorylase from Trypanosoma brucei

Article abstract of DOI:10.1021/cb400411x

Uridine diphosphate N-acetylglucosamine pyrophosphorylase (UAP) catalyzes the final reaction in the biosynthesis of UDP-GlcNAc, an essential metabolite in many organisms including Trypanosoma brucei, the etiological agent of Human African Trypanosomiasis. High-throughput screening of recombinant T. brucei UAP identified a UTP-competitive inhibitor with selectivity over the human counterpart despite the high level of conservation of active site residues. Biophysical characterization of the UAP enzyme kinetics revealed that the human and trypanosome enzymes both display a strictly ordered bi-bi mechanism, but with the order of substrate binding reversed. Structural characterization of the T. brucei UAP-inhibitor complex revealed that the inhibitor binds at an allosteric site absent in the human homologue that prevents the conformational rearrangement required to bind UTP. The identification of a selective inhibitory allosteric binding site in the parasite enzyme has therapeutic potential.

Full text of DOI:10.1021/cb400411x

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A Novel Allosteric Inhibitor of the Uridine Diphosphate
N‑Acetylglucosamine Pyrophosphorylase from Trypanosoma brucei
Michael D. Urbaniak,† Iain T. Collie,† Wenxia Fang,‡ Tonia Aristotelous,† Susanne Eskilsson,†
Olawale G. Raimi,‡ Justin Harrison,† Iva Hopkins Navratilova,† Julie A. Frearson,†
Daan M. F. van Aalten,‡,§ and Michael A. J. Ferguson*,†
†Division of Biological Chemistry and Drug Discovery, ‡Division of Molecular Microbiology, and §MRC Protein Phosphorylation and
Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dundee DD1 5EH, U.K.
S
* Supporting Information
ABSTRACT: Uridine diphosphate N-acetylglucosamine pyro-
phosphorylase (UAP) catalyzes the final reaction in the
biosynthesis of UDP-GlcNAc, an essential metabolite in many
organisms including Trypanosoma brucei, the etiological agent of
Human African Trypanosomiasis. High-throughput screening of
recombinant T. brucei UAP identified a UTP-competitive inhibitor
with selectivity over the human counterpart despite the high level of
conservation of active site residues. Biophysical characterization of the
UAP enzyme kinetics revealed that the human and trypanosome
enzymes both display a strictly ordered bi−bi mechanism, but with
the order of substrate binding reversed. Structural characterization
of the T. brucei UAP−inhibitor complex revealed that the inhibitor
binds at an allosteric site absent in the human homologue that prevents the conformational rearrangement required to bind UTP.
The identification of a selective inhibitory allosteric binding site in the parasite enzyme has therapeutic potential.
he sugar nucleotide uridine diphosphate N-acetylglucos-
infectivity and virulence. Several enzymes involved in the
Tamine (UDP-GlcNAc) is an important and ubiquitous biosynthesis of glycosylphosphatidylinositol anchors3−5 and sugar
nucleotide biosynthesis6−10 have been shown to be essential in
metabolite that is used in eukaryotes as the source of
bloodstream form T. brucei by genetic validation.
N-acetlyglucosamine in the biosynthesis of N-linked and O-linked
glycans and the source of glucosamine in glycosylphosphatidy-
linositol anchors. In addition, UDP-GlcNAc is required for the
formation of lipopolysaccharide and peptidoglycans used in
bacterial cell wall biosynthesis and the formation of chitin for fungal
cell wall biosynthesis. The enzyme UDP-GlcNAc pyrophosphor-
ylase (UAP) is responsible for a key transformation in the bio-
synthesis of UDP-GlcNAc by catalyzing the reversible reaction
between UTP and glucosamine-1-phosphate (Glc-1-P) forming
UDP-GlcNAc and inorganic pyrophosphate (PPi) (Scheme 1). The
enzyme represents a bottleneck between different glycoconjugate
biosynthetic pathways that has the potential to be exploited as a
therapeutic target, provided that species-specific inhibitors can be
found.
Trypanosoma brucei is a protozoan parasite transmitted by the
bite of an infected tsetse fly (Glossina spp.) and is the etiological
agent of Human African Trypanosomiasis (HAT, also known as
African sleeping sickness). The disease is responsible for 10,000
recorded deaths per annum in sub-Saharan Africa, although due
to poor surveillance the true number is estimated to be much
higher.1 Current treatments are expensive, toxic, and difficult to
deliver, leaving an urgent unmet need for improved therapeutic
agents.2 The parasite has a digenetic lifecycle between a
mammalian host and insect vector and produces a complex
array of glycoconjugates, some of which are essential for its
T. brucei UAP (TbUAP) has been genetically validated in
bloodstream form parasites as essential both in vitro and in vivo
and has been proposed as a potential therapeutic target, although
selective inhibition of the parasite UAP would be a therapeutic
requirement.8 Despite the moderate level of overall sequence
similarity between TbUAP and its human counterpart (31%
sequence identity, 50% sequence similarity), only two of the 15
identified substrate-interacting residues in human UAP
(HsUAP) are different,8,11 and there are no known inhibitors
of UAP. In this work we set out to discover novel species-specific
inhibitors of TbUAP through high-throughput screening of the
recombinant enzyme. Through biophysical and structural
characterization, we reveal that the trypanosome and human
UAP differ in the order of sequential substrate binding and that a
primary hit compound is a species-specific UTP-competitive
allosteric inhibitor of TbUAP.
RESULTS AND DISCUSSION
Identification of Novel TbUAP Inhibitors. Recombinant
T. brucei UAP (TbUAP) was screened against a diverse library of
■
Received: June 10, 2013
Accepted: July 8, 2013
Published: July 8, 2013
© 2013 American Chemical Society
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a
Scheme 1. Enzyme-Catalyzed Formation of UDP-GlcNAc
a
The substrates UTP and GlcNAc-1-P are combined to form the
products UDP-GlcNAc and inorganic pyrophosphate. The reaction
can be monitored with pyrophosphatase that converts the inorganic
pyrophosphate to phosphate, and addition of BIOMOL Green reagent
results in an increase in absorption at 650 nm due to the formation of
a malachite green/molybdate complex.
Figure 1. Surface plasmon resonance sensorgrams of TbUAP and
HsUAP binding to substrates. (A) TbUAP binds UTP with a KD of
83.1 0.5 μM, UTP was injected in duplicates at concentrations from
3.9 to 500 μM. The inset shows an equilibrium affinity fit to a 1:1 binding
model. (B) HsUAP does not bind UTP. (C) TbUAP does not bind
GlcNAc-1-P. (D) HsUAP binds GlcNAc-1-P with a complex binding
profile, GlcNAc-1-P was injected in duplicates at concentrations from
9 nM to 20 μM. (E) The ordered sequential binding of substrates to
UAP is reversed between TbUAP (solid arrows) and HsUAP (dashed
arrows).
63,362 molecules using a discontinuous coupled colorimetric
assay that monitors phosphate generation (Scheme 1).8 The
initial 73 hit compounds that showed >25% inhibition at 30 μM
(0.12% hit rate) were triaged by removing compounds that
displayed activity against the E. coli pyrophosphatase coupling
enzyme. The 12 remaining compounds were all confirmed as
TbUAP inhibitors by direct monitoring of their effects on
conversion of the substrate (UTP) to product (UDP-GlcNAc)
by HPLC. Their IC50 values were determined using the coupled
assay. Commercially available analogues of the two most potent
compounds, 1 and 2 (Scheme 2, IC50 = 37 4 and 49 4 μM,
TbUAP and the closest Human homologue UAP-AGX1
(HsUAP) by SPR (Figure 1, Table 1). We found that TbUAP
binds UTP alone with a KD of 83.1 0.5 μM (Km = 26 μM,
Stokes et al.8) but does not bind GlcNAc-1-P alone (Km = 39 μM,
Stokes et al.8). In contrast, HsUAP does not bind UTP alone
(Km = 53 μM, Peneff et al.11), and although it does show
significant binding to GlcNAc-1-P, it was not possible to calculate
an affinity due to complex binding kinetics. These data reveal that
substrate binding to UAPs is strictly ordered but that,
surprisingly, the order of binding is reversed between the two
species. To our knowledge this is the first example of species-
specificity in sequentially ordered bi−bi mechanisms and raises
the intriguing possibility that UTP-competitive inhibitors may
confer species specificity.
Scheme 2. TbUAP Inhibitors Identified by High-Throughput
Screening
Compound 1 Is a Competitive Inhibitor of TbUAP1. To
investigate the mode of inhibition of 1, we examined its binding
to TbUAP and HsUAP by SPR (Figure 2 and Table 1) and found
that it was bound by TbUAP with KD = 2.58 0.07 μM, but that
HsUAP displayed no significant binding (Figure 2A,B). The
binding of 1 to TbUAP was competitive with UTP, with the KD
shifting to KD = 9.30 0.1 μM (IC50 = 37 4 μM) in the
presence of 500 μM of UTP, while the presence of 100 μM of
GlcNAc-1-P did not significantly affect binding with a KD = 2.35
0.03 μM (Figure 2D,C). The selectivity observed by SPR was
confirmed by testing the activity of 1 against TbUAP and HsUAP
in both the coupled assay and HPLC assay, showing consistent
inhibition of TbUAP but no significant inhibition of HsUAP
(Figure 2E-,F). The coupled assay was used to confirm that
inhibition by 1 was competitive with UTP with an apparent Ki of
respectively), were identified by substructure searching, and 30
analogues were purchased and assayed for activity. None of the
compounds showed improved potency over that of the parent
compounds, with the relatively low potencies limiting the
derivation of structure−activity relationships.
TbUAP1 Binds Its Substrates in a Different Order from
HsUAP1. To examine the enzyme reaction mechanism and
explore the mode of action of the most potent inhibitor 1, a series
of surface plasmon resonance (SPR) experiments were employed
(Figure 1 and 2, Table 1). The reaction mechanism of UAP
requires that both UTP and GlcNAc-1-P bind to the enzyme, but
it was unknown if the sequential binding is random or strictly
ordered. We examined the binding of the two substrates to
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first UAP structure from T. brucei or indeed any protist. In
common with other eukaryotic UAP structures,11,12 the TbUAP
structure consists of a central pyrophosphorylase domain of eight
β-strands sandwiched by eight α-helices in a Rossmann fold,13
which contains the active site, flanked by an N-terminal domain
containing the N-terminus (residues 1−62) and additional β-sheets
from the central domain (residues 209−231 and 377−396), and a
short C-terminal domain. Strikingly, the inhibitor binding site is
located away from but facing the active site in a deep hydrophobic
cleft formed by the central and C-terminal domain (Figure 3A,B)
where it is able to form hydrogen bonds between the amide group of
indolin-2-one and the Gly44 carboxyl group at a distance of 2.8 Å
and between the carboxyl group of indolin-2-one with the amide
group of Asp46 at a distance of 2.8 Å, as well as a number of
hydrophobic interactions (Figure 3C,D). In the published structures
of Candida albicans UAP (CaUAP) there is distinct movement in
the N-terminal domain between the apo-form and the GlcNAc-1-P
or UDP-GlcNAc bound forms,12 consistent with an induced-fit
movement that closes the entrance to the binding site upon
substrate binding. The inhibitor makes contact with residues on the
opposite face of a glycine-rich loop that moves to make contact with
the uridine of bound UDP-GlcNAc (Figure 3B), and TbUAP
adopts a conformation that most closely resembles the apo-CaUAP
structure (2YQC, RMSD 2.2 Å) and least resembles the CaUAP
structure with UDP-GlcNAc bound (2YQJ, RMSD 2.7 Å). Thus, 1
appears to act as an allosteric competitive inhibitor of UTP by
stabilizing the N-terminal domain and uridine-binding loop in a
conformation that prevents the binding of UTP yet does not occupy
the UTP binding site itself. Allosteric regulation of TbUAP activity is
consistent with reports that Giardia lamblia UAP activity is altered
in vivo by the allosteric binding of the metabolite glucosamine-6-
phosphate, although in that case binding caused an increase in
activity.14
Compound 1 Structure−Activity Relationships. The
conformation of the allosteric site is such that only the (R)-
enantiomer of 1 can bind, and it is likely that the kinked shape of
the molecule is crucial for its shape-complimentarity to the
pocket. The benzo[1,3]dioxole moiety is deeply buried, making
close contact with Ala397 and Gly232 at the bottom of the cleft
(Figure 3C,D). Consistent with this binding mode, the commercial
structural analogue of 1 that lacks the benzo[1,3]dioxole moiety
does not inhibit TbUAP1, and even replacement of the bridging
methylene with ethylene is not tolerated (Table 2). The indolin-2-
one sits at the top of the cleft, with the unsubstituted edge exposed
to solvent and the methyl and bromide substituents on making
contact with Ala239, Met370, Lys371, and Ala367 (Figure 3C,D).
Removal of the bromine reduces potency >6-fold, and removal of
both the bromine and methyl groups reduces potency ≥10-fold
(Table 2). The observed SAR for the commercial analogues is
consistent with the contacts observed in the crystal structure.
Allosteric Binding Site Is Unique to TbUAP1. Compar-
ison of the TbUAP-1 structure with the structure of HsUAP11
revealed that the central catalytic domains are structurally similar
(RMSD 1.4 Å), but that the flanking N-terminal and C-terminal
domains occupy different positions (maximum Cα atom shift is
9.5 Å, Figure 3E). The inhibitor binding cleft formed by the
central and C-terminal domain is wider (10.3 Å versus 7.7 Å) and
shallower (10.9 Å versus 17.4 Å) due to both significant movement
of the α-helices and nonconservative substitutions. Critically, the
substitution of Gly232 in TbUAP with Asp221 in HsUAP blocks the
benzo[1,3]dioxole binding site, and the substitution of Ala239 in
TbUAP with Arg228 in HsUAP blocks the entrance to the cleft by
Figure 2. Mode of inhibition by compound 1. (A) TbUAP binds 1 with a
KD of 2.58 0.07 μM. (B) HsUAP does not bind 1. (C) The presence of
100 μM Glc-NAc-1-P does not significantly affect the binding of 1 to
TbUAP (KD of 2.35 0.03 μM). Compound 1 was injected in duplicates
at a concentration series of 69 nM to 50 μM. (D) The presence of
500 μM UTP competes for the binding of 1 to TbUAP, increasing the KD
to 9.3 0.1 μM and significantly decreasing binding response (∼3-fold).
Gray lines represent kinetic fit to 1:1 binding model. (E) Inhibition of
TbUAP (closed circles, IC50 = 37 4 μM) and HsUAP (open squares,
IC50 > 1000 μM) in the discontinuous coupled colormetric assay. (F)
Inhibition of TbUAP in the direct HPLC assay (IC50 = 66 8 μM). (G)
Inhibition of TbUAP at varying concentrations of UTP reveals that
inhibition is competitive with UTP with an apparent Ki of 60 μM. The
concentrations of compound 1 used: open circles 300 μM, open squares
30 μM, open triangles 3 μM, and filled circle 0 μM. (H) Inhibition of
TbUAP at varying concentrations of GlcNAc-1-P reveals a mixed mode of
competition (Ki not calculated). The concentrationsof compound1 used:
open circles 300 μM, open squares 100 μM, open triangles 30 μM, and
filled circle 0 μM.
60 μM (Figure 2G), while the mixed mode of inhibition observed
with GlcNAc-1-P did not allow an apparent Ki to be calculated
(Figure 2H).
Compound 1 Binds TbUAP in a Unique Allosteric Site.
To gain further insight into the binding interactions with
TbUAP, cocrystallization with various ligands was attempted. We
were unable to obtain suitable diffraction quality crystals in the
presence of substrates or product, but cocrystallization with 1
alone resulted in crystals that diffracted to high resolution. The
bound complex was refined against synchrotron diffraction data
to 1.75 Å (Table 3), revealing clear density for the inhibitor at a
site distinct from the active site (Figure 3A). This represents the
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a
Table 1. Surface Plasmon Resonance Parameters
ligand(s)
enzyme
ka (M−1 s−1
)
kd (s−1
N/A
ND
)
KD (μM)
Rmax (RU)
UTP
TbUAP
TbUAP
TbUAP
TbUAP
TbUAP
HsUAP
HsUAP
HsUAP
N/A
ND
83.1 0.5
ND
15.20 0.02
ND
GlcNAc-1-P
1
4.13 0.10
2.24 0.04
5.71 0.02
ND
0.11 0.10
0.21 0.03
0.13 0.04
ND
2.58 0.07
9.30 0.09
2.35 0.03
ND
30.67 0.20
10.21 0.05
26.16 0.02
ND
1 + 500 μM UTP
1 + 100 μM GlcNAc-1-P
UTP
GlcNAc-1-P
b
b
b
∼35
ND
1
ND
ND
ND
a
N/A: kinetic parameters are not available due to fast on-rate and off-rate; affinity was determined using equilibrium fit. ND: binding not detected.
No fit was possible due to complex binding kinetics.
b
Figure 3. Structure of TbUAP in complex with 1. (A) Overview of the TbUAP-1 structure (PDB ID 4bqh), which is composed of an N-terminal domain
(beige), a central domain (green) containing the active site (nucleotide binding motif, red), and a C-terminal domain (blue). 1 (light blue) binds at an
allosteric site. (B) 1 forms close contacts with a loop (PGGNG, dark red) between the N-terminal and central domain on the opposite face to the
UDPGlcNAc binding site. The 2F0 − Fc density of 1 is contoured at 1σ (dark blue mesh). Alignment of the TbUAP central domain with the CaUAP
structure 2YQS12 was used to model the UDPGlcNAc position. (C) Binding interactions of 1 with TbUAP, with the 2F0 − Fc density of 1 displayed. (D)
Schematic of the key interactions in the TbUAP-1 complex. (E) C-α trace showing that the allosteric binding site of HsUAP is wider and flatter due to
movement of the N-terminal domain. (F) Binding interactions in the modeled HsUAP-1 complex showing steric clashes produced by nonconservative
substitution. Alignment of the TbUAP central domain with the HsUAP structure 1JV111 (gray) was used to model the binding of 1 (blue, transparent).
forming a salt bridge with Glu44 (Figure 3F). The structural data
thus explain the observed selectivity of 1.
Compound 1 Inhibits Growth in T. brucei Cultures. We
determined that 1 has an EC50 of 30 μM against cultured T. brucei
(data not shown), a surprisingly small drop-off in potency
compared to the IC50 of 30 μM recorded against the recombinant
T. brucei enzyme. To assess the mode of action of 1, we
determined its potency against a TbUAP conditional null mutant
(TbUAP-cKO) cell line in culture. The conditional null mutant,
where both allelic copies of TbUAP are replaced by drug
resistance cassettes, expresses TbUAP from an ectopic copy
under the control of tetracycline.8 The TbUAP-cKO cell line is
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a
GGATCCCTCGAGTCAAATACCA-3′ (BamHI site underlined) and
inserted into pET15b-pp (a modified pET15b with the thrombin site
replaced with PreScission protease) using the NdeI and BamHI RE sites
to give the plasmid pET15b-pp-HsUAP-AX1. Recombinant HsUAP-
His6 was expressed in BL21 (DE3) E. coli from the vector pET15b-pp-
HsUAP-AX1 and purified in a single step using Ni2+ affinity
chromatography using the same condition as reported for TbUAP-
His6.8 The identity of recombinant HsUAP-AX1 was confirmed by
tryptic mass finger printing (Mascot score 1814, 88% sequence
coverage). Purified recombinant UAPs were stored in 50 mM Tris-
HCl pH 7.5, 10 mM MgCl2, and 10% glycerol at −80 °C prior to use.
Expression of TbUAP for Activity Assays. Recombinant TbUAP-
His6 for activity assay was cloned from T. brucei, expressed from the
plasmid pET15b-pp-TbUAP in Escherichia coli BL21 (DE3), and
purified in a single step using Ni2+ affinity chromatography as reported
previously.8 For crystallization trials, the TbUAP gene was cut from the
pET15b-PP-TbUAP1 plasmid and cloned into a BamHI digested
pGEX-6P-1 vector (GE Healthcare). The resulting plasmid, pGEX-6P-
1-TbUAP, encodes a glutathione-S-transferase (GST) fusion TbUAP
separated by a PreScission protease cleavage site.
Table 2. Structures of Selected Commercial Analogues of 1
compd
n
R1
R2
R3
R4
inhibition at 100 μM (%)
1
3
4
5
6
7
1
2
1
1
1
1
Br
H
H
Cl
H
H
Me
Me
Me
H
H
H
H
H
65
0
H
H
27
25
28
13
Cl
Me
H
H
H
Me
Me
H
a
The binding mode of 1 observed in the TbUAP-1 crystal structure is
consistent with the observed structure−activity relationships.
High-Throughput Screening of TbUAP. The TbUAP high-
throughput screen was performed using a Dundee Drug Discovery Unit
in-house diverse compound collection of 63,362 molecules17 against a
discontinuous coupled colorimetric assay. The assay was performed at
RT in 384-well plates in a final reaction volume of 50 μL in reaction
buffer (50 mM Tris pH 7.5, 10 mM MgCl2, 2% v/v glycerol, 1 mM
dithiothreitol, 0.1 mg mL−1 bovine serum albumin, 1 unit mL−1 E. coli
pyrophosphatase) supplemented with 30 μM UTP, 100 μM GlcNAc-1-P,
0.5 nM recombinant TbUAP, and 30 μM test compound with a final
concentration 1% DMSO. Test compounds in 0.5 μL of DMSO were
transferred to the plates prior to the addition of recombinant TbUAP in
24.5 μL of reaction buffer. The reaction was initiated by the addition of the
substrates UTP and GlcNAc-1-P in 25 μL of reaction buffer and allowed
to proceed for 8 min before termination by the addition of 50 μL of
Biomol Green (0.03% malachite green, 0.2% w/v ammonium molybdate,
0.5% Triton X-100 in 0.7 M HCl). The signal was allowed to develop for a
minimum of 30 min before the absorbance of each well was read at
650 nm. The assay gave a robust average Z′ of 0.8 0.1, with an average
coefficient of variance of 1−3% and signal/background of 2.5 0.3 based
on the inclusion of high (uninhibited) and low (no enzyme) control wells
in each of the 183 assay plates.
Compounds with ≥25% inhibition in the screen (100, 0.16% hit rate)
were cherry picked and confirmed by retesting, with a 73% confirmation
rate (Supplementary Table S1). Confirmed hits were tested for potency
against the pyrophosphatase by modifying the discontinuous coupled
colorimetric assay to include 5 mM inorganic pyrophosphate, and
compounds showing >15% difference between the pyrophosphatase
and coupled assay (Supplementary Table S2) were considered TbUAP
hits (12, 0.02% hit rate). The TbUAP hits were repurchased, and 10-point
inhibitor IC50 curves were determined using the discontinuous coupled
colorimetric assay and fitting the dose−response curve to a four-
parameter fit in ActivityBase XE (IDBS).
viable in the presence of tetracycline (permissive conditions), where
expression of TbUAP occurs, although the cellular levels of UDP-
GlcNAc are reduced (16 pmol/1 × 107 cells) compared to the wild
type (80 pmol/1 × 107 cells) due to a reduced level of TbUAP
expression. The EC50 was not significantly changed between the wild-
type and TbUAP-cKO cell line, suggesting that the cytotoxicity of the
compound is not driven by inhibition of TbUAP. To assess whether 1
was able to inhibit TbUAP in T. brucei cells, we treated wild-type cells
with 100 μM 1 (3 × IC50) or DMSO for 3 h and measured the
intracellular levels of sugar nucleotides by LC−MS/MS.15,16 No
significant difference in the level of sugar nucleotides was observed
between the treated and untreated samples. Taken together, these data
suggest that the observed cytotoxicity of 1 against cultured T. brucei is
due to an off-target effect and not through the inhibition of TbUAP.
Concluding Remarks. We have discovered a novel UTP-
competitive inhibitor of T. brucei UAP that displays good selectivity
for the parasite enzyme over the human homologue due to binding
at a previously unidentified allosteric binding site. While the current
inhibitor is of modest potency and the in vivo parasite toxicity is
likely to be due to off-target effects, the structural data will facilitate
the design and synthesis of more potent compounds that may have
therapeutic potential. A potential drawback to the targeting of an
allosteric site rather than the active site is that resistance may occur
more readily due to lack of selective pressure to maintain
interactions with the enzyme substrate. However, as the binding
site is formed by a hinge region between two domains that undergo
induced-fit movement during the catalytic cycle, such substitutions
may not be tolerated.
High pH Anion Exchange Chromatography. The inhibition of
TbUAP and HsUAP was measured using high pH anion exchange
chromatography (HPAEC) to follow the conversion of UTP to UDP-
GlcNAc by TbUAP. The reaction buffer (50 mM Tris pH 7.5, 10 mM
MgCl2, 2% v/v glycerol, 1 mM dithiothreitol, 0.1 mg mL−1 bovine serum
albumin, 1% DMSO) was supplemented with 25 μM UTP, 40 μM
GlcNAc-1-P, and 25 ng TbUAP or 75 ng HsUAP. The reaction (100 μL)
was incubated at 30 °C for 30 min with or without inhibitor, quenched by
the addition of 10 μL of 0.1 M NaOH, and then subjected to HPAEC
chromatography on a CarboPac PA-1 column (Dionex) using conditions
adapted from Tomiya et al.18 The eluent was monitored at 260 nm, and
peaks were assigned by comparison to commercial standards. The IC50
value was calculated using a four-parameter fit of eight-point potency curves
derived from three independent experiments.
Our studies have revealed that the UAP mechanism is strictly
sequentially ordered, but that the order of substrate binding is
reversed between the parasite and human enzyme. As the
parasite UAP strictly binds UTP first, it follows that UTP-
competitive inhibitors may show selectivity for the parasite
enzyme over the human enzyme. Traditional sequence- and
structure-based drug discovery approaches did not predict that
species specificity would be readily achievable due to the high
level of conservation of active site residues, highlighting the
importance of biophysical studies in target evaluation.
METHODS
■
Competition Studies. The kinetic parameters for TbUAP were
determined in the presence of different concentrations of substrates and
inhibitor using the discontinuous coupled colorimetric assay described
above. The reaction was performed either at fixed concentration of
Cloning, Expression, and Purification of HsUAP. Homo sapiens
UAP-AX1 (HsUAP, NP_003106) was amplified by PCR from cDNA
(OriGeneTechnologies) using the primers 5′-GGAATTCCATAT-
GAACATTAATGACCTC-3′ (NdeI site underlined) and 5′-CGC-
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40 μM GlcNAc-1-P and 5−640 μM of UTP in the presence of 0−300 μM
1, or at a fixed concentration of 25 μM UTP and 4−500 μM GlcNAc-1-P
in the presence of 0−300 μM 1, and the data were fitted to the Michaelis−
Menten equation and displayed as a double reciprocal plot. The calculated
apparent Km(UTP)app in the presence of a range of concentrations of 1
was used to calculate Ki by plotting Km(UTP)app against [I] to solve the
equation Kmapp = (Km/Ki)[I] + Km.
Table 3. Details of Diffraction Data Collection and Structure
Refinement for TbUAP1 + 1
a
resolution (Å)
space group
unit cell
25.00 (1.75)
C2221
a (Å)
59.9
Surface Plasmon Resonance. Recombinant TbUAP and HsUAP
were chemically biotinylated and captured on a streptavidin surface
of a Biacore T100 instrument (GE-Healthcare) at densities ∼6,000−
7,000 RU. To stabilize captured proteins over time all experiments were
run at 4 °C. Ligands were injected over captured proteins at flow rate
30 μL min−1 in running buffer (50 mM Tris pH 7.5, 150 mM NaCl, 10 mM
MgCl2, 0.05% Tween 20, 1 mM DTT, 3% DMSO), with each compound
injected in duplicates in concentration series adjusted specifically for each
ligand; UTP was injected at 2-fold concentration series (3.9−500 μM),
GlcNAc-1-P at 3-fold concentration series (9 nM to 20 μM), and 1 at 3-fold
concentration series (69 nM to 500 μM). Association was measured for 30 s
and dissociation for 30−300 s depending on the off-rate. For competition
studies, 500 μM UTP or 100 μM Glc-NAc-1-P was added to the running
buffer. All data were double referenced for blank injections of buffer and
biotin-blocked Streptavidin surface. Scrubber 2 (BioLogic Software) was
used to process and analyze the data.
Expression and Purification of TbUAP for Crystallography.
Recombinant TbUAP-GST was expressed from the plasmid pGEX-6P-
1-TbUAP in E. coli BL21 (DE3) pLysS. Cells were grown in LB at 37 °C
to an OD600 of 0.8 and cooled to RT, and protein expression was
induced with 250 μM isopropyl-β-D-thiogalactopyranoside for 20 h.
Cells were harvested by centrifugation at 3500 × g at 4 °C for 30 min,
resuspended in buffer A (25 mM Tris pH 7.5, 150 mM NaCl) in the
presence of 10 mg mL−1 DNase, a protease inhibitor cocktail (Roche)
and 0.5 mg mL−1 lysozyme), lysed on a EmulsiFlex-C3 homogenizer
at 20 kpsi (Avestin), and centrifuged at 40,000 × g for 30 min. The
supernatant was incubated with prewashed glutathione sepharose beads
(GE Healthcare) at 4 °C on a rotating platform for 2 h, and the beads
were isolated by centrifugation at 1000 × g for 3 min and washed with
buffer A four times. TbUAP was cleaved from the GST tag by treatment
with PreScission protease in the same buffer at 4 °C on a rotating
platform for 18 h, and the released protein was further purified on a
Superdex75 gel filtration column (2.6 cm × 60 cm) (Amersham
Biosciences) with 1.0 mL min−1 buffer A. The fractions were verified by
SDS-PAGE, pooled, and concentrated to 15 mg mL−1 using a 10-kDa
cutoff Vivaspin concentrator (GE Healthcare).
Crystallization, Data Collection, and Structure Determination.
Crystallization was conducted using the sitting-drop vapor diffusion
method at RT, where each drop contained 0.5 μL of TbUAP1 solution
(15 mg mL−1 in buffer A) with an equal volume of the mother liquor. To
obtain the TbUAP1-1 complex, the protein was incubated with 0.495 M
compound at 4 °C for 30 min before setting up crystal trays. The
complex crystallized after 4−5 days in the space group C2221 from a
mother liquor containing 25% PEG3350, 0.2 M (NH4)2SO4, 0.1 M Bis-
Tris pH 5.5. Crystals were cryo-protected in this solution supplemented
with 15% glycerol. X-ray data were collected at the I-24 (microfocus)
beamline of the Diamond (U.K.) synchrotron and processed with
HKL2000.19 The phase problem was solved by the automated molecular
replacement pipeline BALBES;20 REFMAC21 was used for further
refinement and iterated with model building using COOT.22 Detailed
crystallographic parameters are given in Table 3. The model for ligands
was not included until their conformations were fully defined by
unbiased |Fo| − |Fc|, φcalc electron density maps. Ligand structures and
topologies were generated by PRODRG.23 Images were generated with
PyMol24 and LigPlot+.25 The final structure coordinates and structure
factors are available in the PDB (4bqh and r4bqhsf, respectively).
Trypanosoma brucei Growth Inhibition. The potency of 1 against
cultured T. brucei was determined using a standard 3-day Alamar blue
assay as described previously.26 Assays were conducted using the Lister
427 single marker cell line27 or a TbUAP conditional null mutant8 grown
in HMI9-T.28 The EC50 values were calculated from 8-point potency
curves in triplicate.
b (Å)
103.0
c (Å)
187.1
no. of reflections
no. of unique reflections
I/σ (I)
334133
58590
13.6 (4.5)
99.7 (99.2)
5.7 (5.4)
7.3 (34.9)
completeness (%)
redundancy
R
merge (%)
RMSD from ideal geometry
bond dist (Å)
0.01
1.30
18.9
21.9
541
408
bond angle (deg)
R
work (%)
free (%)
R
no. of residues
no. of water molecules
B factors (Å2)
overall
19.5
18.8
13.6
26.9
protein
ligand
water
a
Values in brackets are for the highest resolution shell. All measured
data were included in structure refinement.
Sugar Nucleotide Analysis. T. brucei Lister 427 single marker cells
grown in HMI9-T28 were treated with 100 μM of 1 in 0.1% DMSO or a
0.1% DMSO control for 3 h. Cells were harvested by centrifugation, the
intracellular sugar nucleotides were extracted, and their levels were
quantified by LC−MS/MS analysis as described previously.15,16
ASSOCIATED CONTENT
* Supporting Information
Further details of the 73 initial hits (Table S1) and the 30
commercially available compounds (Table S2). This material is
available free of charge via the Internet at http://pubs.acs.org.
Accession Codes
The TbUAP-1 structure coordinates (4bqh) and structure
factors (r4bqhsf) are available in the PDB.
■
S
AUTHOR INFORMATION
Corresponding Author
*E-mail: m.a.j.ferguson@dundee.ac.uk.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was funded by the Wellcome Trust (Programme
Grant 085622 and 077705, and Strategic Award 083481). This
work was supported by an MRC Program Grant G0900138.
D.M.F.vA. is supported by a Wellcome Trust Senior Research
Fellowship (WT087590MA).
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